Process for the preparation of (L)-2-chloropropionic acid and its salts using lipase from pseudomonas

ABSTRACT

A process for the preparation of (L)-2-chloropropionic acid and its alkali metal, alkaline earth metal or ammonium salts which comprises hydrolyzing isobutyl L-chloropropionate at a pH of from 4 to 8 in the presence of a lipase from Pseudomonas spec. DSM 8246 and isolating the optically active reaction product from the reaction mixture either directly or after conversion of the salt into the acid in a conventional way, or further reacting it in situ. The optically active products are important intermediates for preparing crop protection agents.

The present invention relates to a novel process for preparing(L)-2-chloropropionic acid and its alkali metal, alkaline earth metal orammonium salts.

JP 57 094 295, JP 62 205 797, EP-A 196 625, JP 61 111 699 and Appl.Biochem. Biotechnol. 9(3) (1984) 255 disclose the enantio-selectiveenzymatic cleavage of racemic 2-chloropropionic esters. However, thedisadvantage in this case is, besides the maximum possible yield of only50%, the insufficient enantiomeric purity of the 2-chloropropionatesformed.

Furthermore, EP-A 257 716 discloses a continuous process in which methylD,L-2-bromopropionate is used as racemate and converted in a 2-phasesystem using Candida cylindracea lipase into methylL-(-)-2-bromopropionate. However, no hydrolysis products are isolated inthis process.

Furthermore, S. K. Dahod and P. Sinta-Mangano describe in Biotechnol.Bioeng. 30(8) (1987) 995 the lipase-catalyzed hydrolysis of methylL-2-chloropropionate in the presence of carbon tetrachloride. However,the disadvantage in this case is, besides the use of a chlorinatedsolvent, the low yield of about 30% with an enantiomeric purity of only95%.

In addition, EP-A 511 526 teaches a process for the enzymatic hydrolysisof racemic 2-chloropropionic esters in a 2-phase system which consistsof water and of an organic solvent, which is substantially immisciblewith water, for the ester. This process is very elaborate because it isnecessary several times for the organic phase to be separated off,brought into contact with the hydrolase and recycled again. Since,despite relatively long reaction times, only incomplete conversion takesplace and enantiomerically pure products are not obtained, this methodis unsuitable for the industrial preparation of (L)-2-chloropropionicacid and its sodium salt.

Sodium (L)-2-chloropropionate and (L)-2-chloropropionic acid have todate been prepared by alkaline hydrolysis of isobutyl(L)-2-chloropropionate. However, this often results in a low-qualityproduct (about 90-95% enantiomeric excess) which additionally contains5-10% lactic acid as by-product.

Thus, the object of the present invention was a simple process forpreparing (L)-2-chloropropionic acid and sodium (L)-2-chloropropionateof maximum chemical and optical purity (at least about 98% enantiomericexcess and less than 1% lactic acid).

Accordingly, a process for the preparation of (L)-2-chloropropionic acidand its alkali metal, alkaline earth metal or ammonium salts was foundwhich comprises hydrolyzing isobutyl L-chloropropionate at a pH of from4 to 8 in the presence of a lipase from Pseudomonas spec. DSM 8246*) andisolating the optically active reaction product from the reactionmixture either directly or after conversion of the salt into the acid ina conventional way, or further reacting it in situ. Pseudomonas sp. DSM8246 was deposited with the DSM on Apr. 28, 1993, and was assigned theaccession number DSM 8246 by this International Depository Authority.

Isobutyl L-chloropropionate is advantageously prepared starting fromD-lactic acid which can be prepared, for example, by a biotechnologicalprocess disclosed in EP-A 069 291. This entails a mixture of aqueousglucose solution, yeast autolysate, vitamins, catalytic amounts ofphosphoric acid and a buffer for the lactic acid produced, e.g. calciumcarbonate, being fermented at about 45° C. with the addition of lacticacid bacteria. The pH of the fermentation broth is preferably 4-6. Alactate is produced with evolution of carbon dioxide and is convertedinto D-lactic acid by adding an acid, preferably concentrated aqueoussulfuric acid. The D-lactic acid is subsequently extracted withisobutanol. The resulting D-lactic acid solution is then concentrated.Part of the lactic acid is esterified even during this. The remainingamount of lactic acid is esterified with acid catalysis, sulfuric acidbeing an example of a suitable acid (cf., for example, EP-A 287 426,DE-A 32 14 697 and DE-A 34 33 400). The water formed in the reaction issubsequently removed by distillation together with unreacted isobutanol.

The isobutyl D-lactate obtained can be chlorinated in a conventional waywithout further purification (cf., for example, EP-A 401 104, JP 61 057534 (1986), JP 02 104 560 (1990), JP 61 068 445 (1986) and FR-A 24 59221). The chlorination is preferably carried out with thionyl chloridein the presence of a catalyst, e.g. N,N-dimethylformamide, there beinginversion at the asymmetric carbon atom. High boilers are removed fromthe crude product which is then purified by distillation.

It is possible in the manner described above to obtain isobutylL-chloropropionate with a chemical purity of about 98-99%. The opticalpurity (L:D) is about 98-100%.

The lipase can be obtained from Pseudomonas spec. DSM 8246 bycultivating the bacterium in a nutrient medium and isolating the enzymefrom the culture broth. Suitable nutrient media are those containingcarbon sources, nitrogen sources, inorganic salts and, whereappropriate, small amounts of trace elements and vitamins. Nitrogensources which can be used are inorganic or organic nitrogen compounds ormaterials which contain these compounds. Examples are: ammonium salts,nitrates, corn steep liquor, yeast autolysate, yeast extract andhydrolyzed casein. Carbon sources which can be used are sugars such asglucose, polyols such as glycerol or else organic acids such as citricacid or fatty acids. Particularly suitable carbon sources are vegetableoils such as soybean oil, linseed oil or olive oil. Examples ofinorganic salts are the salts of calcium, magnesium, manganese,potassium, zinc, copper, iron and other metals. Anions of these saltswhich should be particularly mentioned are phosphate and nitrate ions.

Preferred cultivation temperatures are 25° to 33° C. The pH of themedium is kept at 6 to 7.5, preferably at 6.5 to 7, during thefermentation using mineral acids such as 2N sulfuric acid or bases suchas ammonia. The culture is carried out as submerged culture withvigorous aeration and stirring. Fermentation is continued until twoconsecutive measurements of enzyme activity at an interval of threehours show constant activity. An incubation time of from 40 to 60 hoursis generally sufficient. It is possible in this way to obtain enzymeyields of 50 to 500 mg per 1 of culture broth.

The enzyme is isolated from the culture broth in a conventional way. Inorder to separate the microorganisms and insoluble material, the brothis centrifuged or filtered. The lipase is then obtained from thecollected liquid phase either by precipitation with a water-miscibleorganic solvent, e.g. acetone or a lower alcohol, or by adding a salt,especially ammonium sulfate.

To increase the specific activity and to reduce further the content ofimpurities in the resulting crude lipase it is possible to redissolveand then reprecipitate it, for example by adding solvents or salt(fractional precipitation). However, the crude lipase can also bepurified by crossflow filtrations of the enzyme-containing solutionthrough suitable ultrafiltration membranes. In this method, lowmolecular weight impurities pass through the membrane while the enzymeis retained.

The particular advantage of the above process is that the lipaseconcentration in the resulting aqueous solution is very high. It isnormally between about 5 and 30 g/l.

The lipase solution can be used directly for the present process.However, it is also possible to use the lipase immobilized on a solidcarrier. Suitable solid carriers are the inert carrier materialscustomary for this purpose (cf., for example, Enzyme and MicrobialTechnology 14 (1992) 426).

The present process is advantageously carried out in a 2-phase systemcomprising aqueous enzyme solution and isobutyl L-chloropropionatewhich, together with the isobutanol produced in the reaction, forms thesecond phase.

It is also possible to add to the organic phase a solvent which issubstantially immiscible with water, e.g. a hydrocarbon such asn-hexane, a chlorohydrocarbon or an ether.

The hydrolysis of isobutyl L-chloropropionate takes place in a pH rangefrom about 4 to 8, preferably 5 to 7. The pH is normally adjusted beforeadding the lipase and is kept approximately constant during thereaction, normally by adding a base continuously or in portions. It isalso possible, however, to use a suitable buffered system and tointroduce the complete amount of base (e.g. sodium bicarbonate) at theoutset.

Bases suitable for this purpose are, for example, alkali metalhydroxides such as sodium and potassium hydroxides, alkali metal andalkaline earth metal bicarbonates such as sodium bicarbonate, potassiumbicarbonate and calcium bicarbonate, alkali metal and alkaline earthmetal carbonates such as sodium carbonate, potassium carbonate andcalcium carbonate, or tertiary amines such as triethylamine.

Since the preparation of the sodium salt of (L)-2-chloropropionic acidis preferred for reasons of cost, an appropriate sodium compound such assodium hydroxide, sodium bicarbonate and sodium carbonate is used asbase.

The reaction is generally carried out between 5° and 60° C., preferablybetween 20° and 40° C.

The reaction can be carried out under atmospheric pressure, under theautogenous pressure of the reaction mixture or under reduced pressure.

An embodiment which is particularly advantageous in respect of theprogress of the reaction comprises continuous removal of the isobutanolformed during the reaction, preferably by distillation under reducedpressure.

The optically active reaction product can then be isolated from thereaction mixture in a conventional way or be reacted further in situ.Concerning further reaction, reference may be made for example to thestatements in the publications GB-A 20 54 570, DE-A 30 24 265 and EP-A009 285.

In order to isolate the optically active reaction product, the organicphase is separated off together with the lipase. Then the proportion ofisobutanol which is dissolved in the aqueous phase is removed,expediently by distillation (e.g. at 50°-60° C. under 20-50 mbar).Experience has shown that the aqueous sodium (L)-2-chloropropionatesolution then has a residual isobutanol content of less than 0.3% byweight. However, because it cannot be stored at 20°-25° C. for a lengthyperiod, it is advisable for the solution to be cooled until processedfurther or to be acidified and, if desired, worked up to(L)-2-chloropropionic acid. Concentrating the approximately 30% byweight aqueous sodium (L)-2-chloropropionate solution to 50-70% byweight followed by cooling to -20° to -50° C. results, for example, in asuspension of crystals which is stable for several weeks.

The sodium (L)-2-chloropropionate can be isolated in a conventional way,e.g. by the abovementioned low-temperature crystallization, but alsospray- or freeze-drying. In the case of spray-drying, for example withan inlet temperature of 200° C. and an outlet temperature of the solidof 80°-90° C., the sodium (L)-2-chloropropionate is obtained in the formof a dry, free-flowing powder with a residual water content of from 0.2to 0.7%. The optical activity is completely retained in any event.

Acidification of the aqueous sodium (L)-2-chloropropionate solution,e.g. with sulfuric acid, to a pH below 2 results in free(L)-2-chloropropionic acid. The latter can be extracted from the aqueousphase using suitable organic solvents in which 2-chloropropionic aciddissolves well and which have only low miscibility with water. Suitablefor this purpose are, for example, ethers such as methyl tert-butylether or chlorinated hydrocarbons such as dichloromethane and1,2-dichloroethane.

After removal of the organic solvent, the (L)-2-chloropropionic acid canthen be purified in a conventional way, expediently by distillationunder reduced pressure (for example the distillate temperature is about80° C. under 12 mbar).

(L)-2-Chloropropionates and (L)-2-chloropropionic acid are importantintermediates for crop protection agents and drugs. They areparticularly suitable for preparing D-2-phenoxypropionic acid (cf., forexample, DE-A 15 43 841) which can be converted intoR-2-(4-hydroxyphenoxy)propionic acid in a biotechnological process (cf.in this connection, for example, WO 90/11362 and EP-A 465 494).R-2-(4-Hydroxyphenoxy)propionic acid is eventually used as startingmaterial for the preparation of aryloxyphenoxypropionic acid derivativeswith herbicidal activity.

PREPARATION EXAMPLES Example 1

Preparation and purification of the lipase from Pseudomonas spec. DSM8246

The following medium was used to cultivate the microorganism Pseudomonasspec. DSM 8246:

    ______________________________________                                               KH.sub.2 PO.sub.4                                                                            20     g/l                                                     Na.sub.2 HPO.sub.4                                                                           10     g/l                                                     MgSO.sub.4     5      g/l                                                     CaCl.sub.2 × 2H.sub.2 O                                                                3      g/l                                                     FeSO.sub.4 × 7H.sub.2 O                                                                0.5    g/l                                                     MnSO.sub.4 × 4H.sub.2 O                                                                0.005  g/l                                                     CoCl.sub.2 × 6H.sub.2 O                                                                0.005  g/l                                                     CuSO.sub.4 × 5H.sub.2 O                                                                0.005  g/l                                                     ZnSO.sub.4 × 7H.sub.2 O                                                                0.005  g/l                                                     Yeast extract  5      g/l                                              ______________________________________                                    

The carbon source used was refined soybean oil which was pumped in at aconstant rate of 1 g/l×h. The pH was kept constant at pH 6.5 throughoutthe fermentation using 2N H₂ SO₄ and 25% strength NH₄ OH.

The seed culture was obtained by inoculating 400 ml of nutrient brothmedium pH 6.5 with the microorganism Pseudomonas spec. DSM 8246.

The seed culture was incubated on a shaker at 30° C. for 10 h.

The medium was inoculated at 30° C. and pH 6.5 with 5 parts by volume ofthe seed culture per 100 parts by volume of medium. The main cultivationwas carried out at 30° C. in 10 l stirred fermenters with a content of 8l. The stirring speed of the inbuilt paddle stirrers was 1000revolutions per minute, and the aeration rate was one volume of air perminute and volume of fermentation broth. After 60 h, the fermentationbroth showed a constant activity in two consecutive activitymeasurements of 300 F.I.P. (=Federation International Pharmaceutique)enzyme units per ml (for the method, see, for example, R. Ruyssen and A.Lauwers, Pharmaceutical Enzymes, E. Story-Scientia P.V.B.A., ScientificPublishing Company, Gent/Belgium, 1978, pages 78-82).

The fermentation was then stopped and the lipase which was produced wasisolated from the fermentation broth in the following way:

The discharge from the fermenter was diluted with n-propanol to 65% byvolume alcohol. The biomass and precipitated by-products were removed bycentrifugation. The clear, alcoholic enzyme solution was concentratedunder reduced pressure to one third of the initial volume. Although thisenzyme solution was already suitable for the hydrolysis of isobutylL-chloropropionate, it was washed with three volumes of water in adiafiltration unit (cellulose triacetate crossflow filtration units,separation limit 20,000 nominal molecular weight, from Sartorius,Gottingen) to increase the activity further and then concentrated to onequarter of the initial volume by filtration. The lipase was precipitatedfrom this enzyme concentrate by adding n-propanol to a content of 85% byvolume. The precipitate containing the lipase activity was harvested bycentrifugation and taken up in an aqueous solution containing 65 partsby volume of n-propanol. The ratio by weight of precipitate ton-propanol/water mixture was 1 to 10.

Undissolved precipitate was removed by centrifugation. The lipase wasprecipitated from the clear supernatant from the centrifugation byincreasing the n-propanol content to 80 parts by volume. The precipitatewas harvested by centrifugation and was freeze-dried. The enzyme powderobtained in this way had a specific activity of 7100 F.I.P. enzyme unitsper milligram of protein.

Example 2

Hydrolysis of isobutyl L-chloropropionate using lipase (according to theinvention)

A suspension of 200 g of isobutyl L-chloropropionate in 400 g of waterwas vigorously stirred at 20°-25° C. and neutralized with 25% by weightaqueous sodium hydroxide solution (pH=7.5). Then 500 mg of lipase (fromPseudomonas spec. DSM 8246; activity about 400 U/mg) were added and thepH of the reaction mixture was kept constant at 7.5 by continuousaddition of 10 normal aqueous sodium hydroxide solution. After 61/4 h,97.3% of the theoretically required amount of sodium hydroxide solutionhad been consumed. In order to stop the reaction, the isobutanol phasewas removed together with the lipase. Freeze-drying of the aqueous phaseresulted in sodium (L)-2-chloropropionate with an enantiomeric purity of99.2%. Yield: 95.8%. The contamination with lactic acid was below 0.05%.

Example 3

Hydrolysis of isobutyl L-chloropropionate using immobilized lipase(according to the invention):

A suspension of 1000 g (6.08 mol) of isobutyl L-chloropropionate in 2000g of water was vigorously stirred at 20°-25° C. and 25% by weightaqueous sodium hydroxide solution was added until the pH was 5-6. Then2.5 g of lipase (activity about 400 U/mg; from Pseudomonas spec. DSM8246; lipase immobilized on "Accurel® EP100" polypropylene powder fromAkzo, particle size 200-1000μ) were added, keeping the pH of thereaction mixture constant (between 5 and 6) by continuous addition of25% by weight sodium hydroxide solution. After 98% of the theoreticallyrequired amount of sodium hydroxide solution had been consumed (afterabout 18 h), the lipase was filtered off. The organic phase of thefiltrate was separated off. Isobutanol dissolved in the aqueous phasewas removed by distillation as azeotrope with water at 60° C. under 50mbar in a thin-film evaporator. 2340 g of an approximately 30% by weightsolution of sodium L-chloropropionate in water were obtained (yield:about 88%).

450 g of this solution were acidified to a pH of about 1 with about 60 gof concentrated sulfuric acid. After addition of about 200 ml of methyltert-butyl ether the organic phase was separated off. The aqueous phasewas extracted three times with 200 ml of methyl tert-butyl ether eachtime. The combined organic phases were dried over sodium sulfate andconcentrated at 300 mbar/50° C., after which the residue was distilledthrough a 30 cm Vigreux column. 84 g of L-chloropropionic acid wereobtained as a colorless liquid of boiling point 82° C./13 mbar with achemical purity of more than 99% and an optical purity of L:D=99:1.

1500 g of the above solution of sodium L-chloropropionate in water werespray-dried in a tower with an inlet temperature of 200° C. At an outlettemperature of 83° C., 480 g of solid sodium L-chloropropionate wereobtained as a white powder with a residual water content of 0.4% and anoptical purity of 99:1 (L:D).

Example 4

Hydrolysis of isobutyl L-chloropropionate using lipase with continuousremoval of isobutanol (according to the invention)

A mixture of 164.5 g (1.0 mol) of isobutyl L-chloropropionate and 330 gof water was stirred at 35°-40° C. while 25% by weight aqueous sodiumhydroxide solution was added until the pH was 5-6. 5.0 ml of anapproximately 5% by weight lipase solution (activity about 100,000 U/ml)were then added to the mixture. The reaction took place at 35°-40° C.while keeping the pH of the reaction mixture at 5-6 by metering in 25%by weight sodium hydroxide solution. After about 20% of the calculatedamount of sodium hydroxide solution had been consumed, the pressure wasslowly reduced to 80 mbar, until the reaction mixture boiled. Thecolorless distillate with a boiling point of 37° C. under 80 mbarcomprised water, isobutanol and small amounts of isobutylL-chloropropionate*). Complete conversion of the initial ester (afterabout 2 hours) was evident from a large rise in the pH of the reactionmixture after each further drop of sodium hydroxide solution. Additionof sodium hydroxide solution was then stopped immediately. The mixturewas then stirred for a few minutes until the pH had returned to about 6.507 g of a 23% strength sodium L-2-chloropropionate solution containingless than 0.5% isobutanol and not more than 0.05% isobutylL-chloropropionate were obtained. Yield: 89% (based on isobutylL-chloropropionate employed).

Example 5 (=Comparative example)

Hydrolysis of isobutyl (L)-2-chloropropionate with sodium hydroxidesolution (without lipase)

2467 g (15 mol) of IB-L-C (enantiomeric purity at least 99%) and 1.6 lof water were placed in a 6 l jacketed reaction vessel and stirred. Theresulting suspension had a pH of about 2 and, at 40°-45° C., a total of1200 g (15 mol) of 50% by weight aqueous sodium hydroxide solution wasadded. The addition took place in accordance with the consumption ofsodium hydroxide solution so that the pH remained constant at 12.3.After the addition was complete (about 2 hours) the reaction mixture wasneutralized (pH=7-8) with 20% by weight hydrochloric acid. An azeotropeof isobutanol and water was then removed by distillation at about 40° C.under 60 mbar, approximately 500 ml of water being additionallyintroduced into the distillation vessel during the distillation. Afterabout 100 min, about 2000 g of azeotrope had distilled out. The residuewas worked up to the product in a conventional way. Yield: about 96%;optical purity: 95.5:4.5 (L:D).

We claim:
 1. A process for the preparation of (L)-2-chloropropionic acidor its alkali metal salt, alkaline earth metal salt or ammonium saltwhich comprises hydrolyzing isobutyl (L)-2-chloropropionate at a pH offrom 4 to 8 with a lipase from Pseudomonas spec. DSM 8246 and isolating(L)-2-chloropropionic acid or its salt from the reaction mixtureconsisting of an organic phase and an aqueous phase either directly orafter conversion of the salt into the acid in a conventional way, orfurther reacting the (L)-2-chloropropionic acid or its salt in situ. 2.The process as claimed in claim 1, wherein the reaction is carried outat from 5° to 60° C.
 3. The process as claimed in claim 1, wherein thepH range is kept constant during the reaction by adding a base.
 4. Theprocess as claimed in claim 1, wherein the reaction is carried out withcontinuous removal of the isobutanol produced thereby.
 5. The process asclaimed in claim 1, wherein the lipase is immobilized on a solidcarrier.
 6. The process according to claim 1, wherein(L)-2-chloropropionic acid or its salt is isolated from the reactionmixture bya) removing the lipase and the organic phase, b) removingdissolved isobutanol from the aqueous phase and c) isolating the(L)-2-chloropropionic acid by transferring it into an inert organicsolvent and isolating it therefrom.